1. Field of the Invention
The present invention relates to detection methods, and more particularly, to the use of metallic surfaces to enhance intensity of fluorescence species or reactions in capture assays thereby increasing the sensitivity and rapidity of these assays. The present invention is applicable for determining free unbound bilirubin in serum and for capturing nucleotide sequences.
2. Background of the Related Art
Assays are used widely for the detection and determination of a variety of proteins, peptides and small molecules. Currently, there exists a large diverse family of assays today and the basic principles are generally the same. These assays typically use receptor-ligand binding for target molecule recognition and fluorescence based readouts for signal transduction. Fluorescent based assay systems are available in many forms, such as time-resolved assays, energy transfer assays and fluorescence polarization assays.
Fluorescence detection is the basis of most assays used in drug discovery and high throughput screening (HTS) today. In all of these assays, assay rapidity and sensitivity is a primary concern. The sensitivity is determined by both the quantum yield of the fluorophores and efficiency of the detection system, while rapidity is determined by the physical and biophysical parameters of temperature, concentration, assay bioaffinity, etc.
Heretofore, assay methods and/or systems have been lacking in sensitivity for determining and quantifying the amount of free unbound bilirubin in neonatal serum or isolating target nucleotide sequence.
Technology has been developed that recognizes that close-proximity to noble metallic surfaces can alter the radioactive decay rate and/or excitation rate of fluorophores. Further, it has been shown that quantum yield of low quantum yield fluorophores can be increased by proximity to such metallic surfaces. However, the use of such technology, termed Metal Enhance Fluorescence (MEF), has been limited and heretofore has not been envisioned for the use of determining the level of free unbound bilirubin in neonatal serum or for isolating a desired nucleotide sequence.
The most commonly used method for serum free-bilirubin measurement is the peroxidase method. The concentration of unbound bilirubin is determined from the peroxidase-catalyzed oxidation of bilirubin by a peroxide [47]. The protocol for measurement of free bilirubin according to the peroxidase method requires a blood sample to be drawn from the baby. The serum, the portion of the sample to be tested, is then separated by centrifugation. The serum is taken on ice and shielded from the light, and is used to measure free bilirubin using the unbound bilirubin UB Analyzer, a direct free bilirubin measurement. The UB Analyzer (FDA approved) in essence utilizes the peroxidase method, but in a standardized instrument. First, a measurement is performed using the full concentration of the peroxidase enzyme, and a readout is obtained which indicates both total and free bilirubin levels. A second measurement is performed using half the initial concentration of peroxidase. To improve the accuracy of the free bilirubin measurement, both the readouts are used to derive the final estimated value of free bilirubin using a known algorithm table.
However, the UB Analyzer has some technical pitfalls including the need for reagent manipulation and sample dilution before analysis. A 40-fold dilution must be made to the serum sample, which can alter intrinsic bilirubin binding properties and mask the presence of binding competitors to albumin. Moreover, there is a possibility of interference with free bilirubin measurement by direct or conjugated bilirubin [48]. The test also requires the use of at least two peroxidase concentrations in order to improve the accuracy of the free bilirubin measurement, as an estimate of the equilibrium free bilirubin in the sample being measured. This necessary and repeated measurement with two different peroxidase concentrations increases both the amount of blood and time required for each sample. Furthermore, the light absorption of bilirubin varies with the type of albumin present and the number of bilirubin molecules bound per albumin. There are also factors that can cause the overestimation or underestimation of the free bilirubin measurement, depending on the rate of the peroxidase reaction [49].
There are also several other cumbersome techniques that indirectly measure unbound bilirubin. For example, the HBABA method, utilizes 2-(4′-hydroxybenzeneazo) benzoic acid to measure the available albumin binding sites of a sample, by a shift in the absorbance spectrum of the dye when bound to albumin [50]. This gives an estimate of how much bilirubin is unbound. The fluorescence-quenching method allows the determination of the binding capacity and affinity of albumin, whereby the concentration of unbound bilirubin may be indirectly calculated, based on the quenching of the ultraviolet fluorescence of albumin upon binding to bilirubin [51].
Providing a sensitive and reliable assay for determining serum free bilirubin would be of great value because jaundice (unconjugated hyperbilirubinemia) is one of the most common problems of prematurity. Almost all premature babies have some degree of jaundice during their first week. Jaundice can lead to neurotoxicity including deafness, auditory neuropathy, athetoid cerebral palsy, supranuclear gaze palsy, neonatal seizures, and apnea [31-33]. Premature infants are at a higher risk of bilirubin-induced neuronal injury than term infants [34]. To prevent bilirubin-induced neurotoxcity, neonates are often treated with intensive phototherapy. In rare cases with severe hyperbilirubinemia and unresponsiveness to phototherapy, exchange transfusion is used. Uniform guidelines, however, do not exist for the management of unconjugated hyperbilirubinemia in premature infants. Currently, serum total bilirubin levels are used to evaluate and manage premature infants with unconjugated hyperbilirubinemia. However, there is substantial evidence that serum total bilirubin levels correlate poorly with bilirubin-induced neurotoxicity in premature infants [35-37]. Moreover, institutional variations in the levels of bilirubin at which phototherapy and exchange transfusions are initiated in jaundiced premature newborns indicate that the current management of hyperbilirubinemia in these babies is not evidence based [38].
Various biochemical factors are involved in the pathogenesis of bilirubin encephalopathy. Bilirubin binding is a complex function of the concentrations of total bilirubin, free unbound bilirubin and serum albumin. According to current theory, unbound bilirubin (UB; also referred to as non-albumin-bound or free bilirubin) is capable of crossing the intact blood brain barrier and causing subsequent neuronal damage [39]. Current literature supports the notion that the risk of bilirubin neurotoxicity increases with increasing free bilirubin (or UB) concentration. According to “free bilirubin thinking,” the free bilirubin concentration determines the distribution of bilirubin between the tissues and vascular space [40]. There exists overwhelming clinical evidence to support this free bilirubin theory [41-46]. Studies in neonates supporting free bilirubin theory have involved autopsy findings of kernicterus, and auditory brainstem response (ABR) findings of transient bilirubin encephalopathy. The findings of these studies have suggested that the neurological outcome of hyperbilirubinemia correlate better with free bilirubin than total serum bilirubin levels. In premature infants, overt kernicterus becomes likely with unbound bilirubin levels ≧15 nmol/L (0.87 μg/dl) [42-43], and ABR changes are seen at unbound bilirubin levels >0.5 μg/dl [41]. In term neonates, ABR changes are seen at unbound bilirubin levels >1.0 μg/dl [45]. In summary, as far as the available biochemical measures are concerned, most of the published studies indicate that free bilirubin is the most sensitive biochemical measure to evaluate premature infants with jaundice.
Due to the shortcomings of the techniques discussed above, it would be advantageous to have a system for measuring unbound bilirubin that not only directly measures the metal-amplified fluorescence of the unbound bilirubin itself but also provides a direct correlation between the fluorescence emission and the concentration of the free bilirubin, even in whole unseparated blood.
Notably, the present invention also addresses the problems relating to isolation and quantitation of specific nucleotide sequences, such as RNA molecules, from biological samples. Isolating and determining a specific nucleotide sequence is an essential tool for the study of regulated gene expression [119] and is routinely employed in studies of gene transcription, [120] RNA stability, [121] RNA transport and a host of other biological processes [122]. In addition, RNA detection and quantitation also present an appealing strategy for rapidly identifying unknown biological agents (bacterial, viral, etc.) [123, 124]. Furthermore, nucleotide sequence detection is of great utility for gene expression profiling in clinical settings, where the expression of a subset of genes within tissue (i.e. biopsy) or blood samples may be rapidly measured, revealing diagnostic information to direct patient-specific therapeutic strategies [120, 125].
All current techniques for quantifying specific RNAs exploit base-pair complimentarity between a target RNA and one or more nucleic acid probes, either in the form of extended DNA or RNA sequences including Northern blots, [119]; RNase protection assays, [126, 127]; [RPAs]) or short oligonucleotides (reverse transcription-PCR [RT-PCR], [128]; or RNA capture assays [129]. This principle allows for extremely precise target recognition, yet current methods of probe:target hybrid detection face a number of technological restrictions. In particular, the utility of RNA sensing in microbial detection and/or clinical gene expression profiling may be hindered by two principal constraints, namely: sensitivity and rapidity [130].
RNA capture assays offer a simple and rapid approach to RNA quantitation. Target RNAs are selected based on complimentarity to an oligonucleotide probe which is attached to a solid surface or matrix, then detected by annealing a radio- or chemically-labeled probe at a distinct site on the target RNA [129]. At present, however, these assays are subject to the same sensitivity limitations as those described for Northern blots and RPAs, namely, that detection relies on the activity of radiolabels, the sensitivity of conjugated fluorophores, or the use of bright secondary chemiluminescent assays. These conditions make RNA capture assays currently useful only for abundant RNA species, thus limiting their general utility as a biosensor platform [128].
Thus, there is a need for biosensor systems and methods of using same that overcome the shortcomings of the prior art and provide for increased sensitivity and signal production for use in determining free bilirubin in blood or serum, and isolating target nucleotide sequences.